2.1. Gene Expression
Every living organism is a product of expression of its genes in response to a developmental program (encoded in the genome itself) and environmental factors. Gene expression can be defined as the conversion of the nucleotide sequence of a gene into the amino acid sequence of a protein or into the nucleotide sequence of a stable RNA.
In eukaryotes, gene expression begins in the nucleus with the transcription of a gene into a premessenger-RNA, also referred to as a primary transcript. While still in the nucleus, the pre-mRNA is extensively modified. Each primary transcript is capped at the 5′ end, associates with hnRNP proteins to form messenger RNA-protein particles (“mRNPs”), acquires a polyadenylic acid tail at the 3′ end, and undergoes splicing to remove introns. In addition, the nucleotide sequence of certain pre-mRNAs can be altered post-transcriptionally in a process known as RNA editing. Thus processed, the mature mRNA is exported to the cytoplasm. Upon export, mRNA dissociates from hnRNP proteins and binds a set of cytosol-specific mRNA-binding proteins. Once in the cytoplasm, the mRNA either immediately associates with ribosomes and templates for protein synthesis or is localized to discrete cellular foci to direct compartment-specific protein synthesis. Degradation of mRNA and protein, which occurs both in the nucleus and the cytoplasm, concludes the list of processes that comprise gene expression.
2.2. Post-Transcriptional Gene Expression Regulation
Gene expression is very tightly regulated. To produce the desired phenotype, each gene must be expressed at a defined time and at a defined rate and amount. Extensive experimental evidence indicates that post-transcriptional processes such as mRNA decay, translation, and mRNA localization constitute major control points in gene expression.
An aberration in the expression of one or more genes can be the cause or a downstream effect of a disease or other abnormality. Understanding gene expression regulation mechanisms in the normal/healthy/wild-type cell/body and during pathology will permit rational therapeutic intervention.
Regulation of gene expression both at the mRNA stability and translation levels is important in cellular responses to development or environmental stimuli such as nutrient levels, cytokines, hormones, and temperature shifts, as well as environmental stresses like hypoxia, hypocalcemia, viral infection, and tissue injury (reviewed in Guhaniyogi & Brewer, 2001, Gene 265(1-2):11-23). Furthermore, alterations in mRNA stability have been causally connected to specific disorders, such as neoplasia, thalassemia, and Alzheimer's disease, (reviewed in Guhaniyogi & Brewer, 2001, Gene 265(1-2):11-23 and Translational Control of Gene Expression, Sonenberg, Hershey, and Mathews, eds., 2000, CSHL Press). In contrast, regulation of gene expression at the mRNA localization level is primarily used by the cell to create and maintain polarity (internal gradients of protein concentration) (reviewed in Translational Control of Gene Expression, Sonenberg, Hershey, and Mathews, eds., 2000, CSHL Press).
2.3. mRNA Untranslated Regions in Gene Expression Regulation
A typical mRNA contains a 5′ cap, a 5′ untranslated region (“5′ UTR”) upstream of a start codon, an open reading frame, also referred to as coding sequence, that encodes a stable RNA or a functional protein, a 3′ untranslated region (“3′ UTR”) downstream of the termination codon, and a poly(A) tail. Most studied cis-dependent RNA-based gene expression regulation elements map to the 5′ or 3′ UTRs.
Examples of 5′ UTR regulatory elements include the iron response element (“IRE”), internal ribosome entry site (“IRES”), upstream open reading frame (“uORF”), male specific lethal element (“MSL-2”), G-quartet element, and 5′-terminal oligopyrimidine tract (“TOP”) (reviewed in Keene & Tenenbaum, 2002, Mol Cell 9:1161 and Translational Control of Gene Expression, Sonenberg, Hershey, and Mathews, eds., 2000, CSHL Press).
Examples of 3′ UTR regulatory elements include AU-rich elements (“AREs”), Selenocysteine insertion sequence (“SECIS”), histone stem loop, cytoplasmic polyadenylation elements (“CPEs”), nanos translational control element, amyloid precursor protein element (“APP”), translational regulation element (“TGE”)/direct repeat element (“DRE”), bruno element (“BRE”), 15-lipoxygenase differentiation control element (15-LOX-DICE), and G-quartet element (reviewed in Keene & Tenenbaum, 2002, Mol Cell 9:1161).
The internal ribosome entry site (“IRES”) is one of the 5′ UTR-based cis-acting elements of post-transcriptional gene expression control. IRESes facilitate cap-independent translation initiation by recruiting ribosomes directly to the mRNA start codon. IRESes are commonly located in the 3′ region of a 5′ UTR and are, as recent work has established, frequently composed of several discrete sequences. IRESes do not share significant primary structure homology, but do form distinct RNA secondary and tertiary structures. Some IRESes contain sequences complementary to 18S RNA and therefore may form stable complexes with the 40S ribosomal subunit and initiate assembly of translationally competent complexes. A classic example of an “RNA-only” IRES is the internal ribosome entry site from Hepatitis C virus. However, most known IRESes require protein co-factors for activity. More than 10 IRES trans-acting factors (“ITAFs”) have been identified so far. In addition, all canonical translation initiation factors, with the sole exception of 5′ end cap-binding eIF4E, have been shown to participate in IRES-mediated translation initiation (reviewed in Vagner et al., 2001, EMBO reports 2:893 and Translational Control of Gene Expression, Sonenberg, Hershey, and Mathews, eds., 2000, CSHL Press).
AU-rich elements (“AREs”) are 3′ UTR-based regulatory signals. AREs are the primary determinant of mRNA stability and one of the key determinants of mRNA translation initiation efficiency. A typical ARE is 50 to 150 nucleotides long and contains 3 to 6 copies of AU3A pentamers embebbed in a generally A/U-enriched RNA region. The AU3A pentamers can be scattered within the region or can stagger or even overlap (see, e.g., Chen et al., 1995, Trends Biol Sci 20:465). One or several AU3A pentamers can be replaced by expanded versions such as AU4A or AU5A heptamers (see, e.g., Wilkund et al., 2002, J Biol Chem 277:40462 and Tholanikunnel and Malborn, 1997, J Biol Chem 272:11471). Single copies of the AUnA (where n=3, 4, or 5) elements placed in a random sequence context are inactive. The minimal active ARE has the sequence U2AUnA(U/A)(U/A) (where n=3, 4, or 5) (see, e.g. Worthington et al., 2002, J Biol Chem, 277:48558-64). The activity of certain AU-rich elements in promoting mRNA degradation is enhanced in the presence of distal uridine-rich sequences. These U-rich elements do not affect mRNA stability when present alone and thus that have been termed “ARE enhancers” (see, e.g., Chen et al., 1994, Mol. Cell. Biol. 14:416).
Most AREs function in mRNA decay regulation and translation initiation regulation by interacting with specific ARE-binding proteins (“AUBPs”). There are at least 14 known cellular proteins that bind to AU-rich elements. AUBP functional properties determine ARE involvement in one or both pathways. For example, ELAV/HuR binding to c-fox ARE inhibits c-fos mRNA decay (see, e.g., Brennan & Steitz, 2001, Cell Mol Life Sci. 58:266), association of tristetraprolin with TNFα ARE dramatically enhances TNFα mRNA hydrolysis (see, e.g., Carballo et al., 1998, Science 281:1001), whereas interaction of TIA-1 with the TNFα ARE does not alter the TNFα mRNA stability but inhibits TNFα translation (see, e.g., Piecyk et al., 2000, EMBO J. 19:4154). Given its size, it is very likely that one copy of a typical ARE is capable of interacting with several AUBPs molecules. Therefore, it is contended that in the cell the competition of multiple AUBPs for the limited set of AUBP-binding sites in an ARE and the resulting “ARE proteome” determines the ARE regulatory output.
The mechanism of ARE-mediated mRNA decay is poorly understood. It has been established that mammalian mRNA degradation proceeds in 3′ to 5′ direction and that the first step is deadenylation by poly(A)-specific ribonuclease (“PARN”). Recent work indicates that following deadenylation a stable multi-ribonuclease complex, termed exosome, degrades the body of the message. Exosome alone is capable of initiating and accomplishing mRNA decay. However, the presence of certain AREs upregulates degradation efficiency. Available evidence suggests that AREs alone or bound by AUBPs help recruit exosome to the RNA (see, e.g., Chen et al., 2001, Cell 107:451 and Mukherjee et al., 2002, EMBO J. 21:165).
It has been reported that degradation of some mRNAs depends on ongoing translation. Thus, the translation machinery can also serve as a ribonulease-recruiting or stabilizing AUBPs-removing entity. Supporting evidence indicates that this mechanism may operate only on a subset of mRNAs under special cell growth conditions (see, e.g., Curatola et al., 1995, Mol. Cell. Biol. 15:6331; Chen et al., 1995, Mol. Cell. Biol. 15:5777; Koeller et al., 1991, Proc. Natl. Acad. Sci. 88:7778; Savant-Bhonsale et al., 1992, Genes Dev. 6:1927; and Aharon & Schneider, 1993, Mol. Cell. Biol. 13:1971).
The mechanism of ARE-dependent translation regulation is understood even less well than that of ARE-mediated mRNA decay. It is not clear how a 3′ UTR-localized element can affect translation initiation, a process that takes place in the 5′ UTR. One plausible explanation comes from recent work showing that most or all cytoplasmic mRNPs are circularized via eIF4F—poly(A)-binding protein (“PABP”) interaction. This interaction can bring AREs in the 3′ UTR into close proximity to the translation initiation site (see, e.g., Wells et al., 1998, Mol. Cell. 2:135).
Citation or identification of any reference in Section 2 of this application is not an admission that such reference is available as prior art to the present invention.